Without new methods for biofuel production, the world will continue to depend on fossil fuels for transportation. Accelerating demand, diminishing reserves and geopolitical risks have in recent years dramatically driven up the cost of fossil fuels. Use of fossil fuels also releases carbon dioxide into the atmosphere, which may cause deleterious environmental effects. Many governments have prescribed a reduction in the use of fossil fuels in favor of alternative renewable biofuels in an effort to stem the release of carbon dioxide from transportation vehicles.
Ethanol can be used as renewable biofuel but methods do not currently exist that can produce ethanol in sufficient quantities and at a price that could lead to a widespread adoption of ethanol as a major alternative to fossil fuels in the worldwide transportation fuel market.
The patent and scientific literature cited herein establishes the knowledge that is available to those with skill in the art. The issued U.S. and foreign patents, published U.S. and foreign patent applications, and all other publications cited herein are hereby incorporated by reference. Additionally, all amino acid and nucleic acid sequences with the respective amino acid sequences encoded thereby identified by database accession number are hereby incorporated by reference.
Aspects of the invention utilize techniques and methods common to the fields of molecular biology, microbiology and cell culture. Useful laboratory references for these types of methodologies are readily available to those skilled in the art. See, for example, Molecular Cloning: A Laboratory Manual (Third Edition), Sambrook, J., et al. (2001) Cold Spring Harbor Laboratory Press; Current Protocols in Microbiology (2007) Edited by Coico, R, et al., John Wiley and Sons, Inc.; The Molecular Biology of Cyanobacteria (1994) Donald Bryant (Ed.), Springer Netherlands; Handbook Of Microalgal Culture: Biotechnology And Applied Phycology (2003) Richmond, A.; (ed.), Blackwell Publishing; and “The cyanobacteria, molecular Biology, Genomics and Evolution”, Edited by Antonia Herrero and Enrique Flores, Caister Academic Press, Norfolk, UK, 2008.
Definitions
As used herein, the term “metabolically enhanced” refers to any change in the endogenous genome of a wild type cell or to the addition of non-endogenous genetic code to a wild type cell, e.g., the introduction of a heterologous gene. More specifically, such changes are made by the hand of man through the use of recombinant DNA technology or mutagenesis. The changes can involve protein coding sequences or non-protein coding sequences such as regulatory sequences as promoters or enhancers.
The term “nucleic acid” is intended to include nucleic acid molecules, e.g., polynucleotides which include an open reading frame encoding a polypeptide, and can further include non-coding regulatory sequences, and introns in the case of eukaryotic organisms for example algae. In addition, the terms are intended to include one or more genes that map to a functional locus. In addition, the terms are intended to include a specific gene for a selected purpose. The gene can be endogenous to the host cell or can be recombinantly introduced into the host cell.
The phrase “operably linked” means that the nucleotide sequence of the nucleic acid molecule or gene of interest is linked to the regulatory sequence(s) in a manner which allows for expression (e.g., enhanced, increased, constitutive, basal, attenuated, decreased or repressed expression) of the nucleotide sequence and expression of a gene product encoded by the nucleotide sequence (e.g., when the recombinant nucleic acid molecule is included in a recombinant vector, as defined herein, and is introduced into a microorganism).
The term “recombinant nucleic acid molecule” includes a nucleic acid molecule (e.g., a DNA molecule) that has been altered, enhanced or engineered such that it differs in nucleotide sequence from the native or natural nucleic acid molecule from which the recombinant nucleic acid molecule was derived (e.g., by addition, deletion or substitution of one or more nucleotides). Advantageously, a recombinant nucleic acid molecule (e.g., a recombinant DNA molecule) includes an isolated nucleic acid molecule or gene of the present invention
The terms “host cell” and “recombinant host cell” are intended to include a cell suitable for metabolic manipulation, e.g., which can incorporate heterologous polynucleotide sequences, e.g., which can be transformed. The cell can be a prokaryotic or a eukaryotic cell. The term is intended to include progeny of the cell originally transformed. In particular embodiments, the cell is a prokaryotic cell, e.g., a cyanobacterial cell. Particularly, the term recombinant host cell is intended to include a cell that has already been selected or engineered to have certain desirable properties and suitable for further enhancement using the compositions and methods of the invention.
The term “promoter” is intended to include a polynucleotide segment that can transcriptionally control a gene-of-interest, e.g., a pyruvate decarboxylate gene that it does or does not transciptionally control in nature. In one embodiment, the transcriptional control of a promoter results in an increase in expression of the gene-of-interest. In another embodiment, a promoter is placed 5′ to the gene-of-interest. A promoter can be used to replace the natural promoter, or can be used in addition to the natural promoter. A promoter can be endogenous with regard to the host cell in which it is used or it can be a heterologous polynucleotide sequence introduced into the host cell, e.g., exogenous with regard to the host cell in which it is used. Promoters of the invention may also be inducible, meaning that certain exogenous stimuli (e.g., nutrient starvation, heat shock, mechanical stress, light exposure, etc.) will induce the promoter leading to the transcription of the gene behind.
The term “about” is used herein to mean approximately, in the region of, roughly, or around. When the term “about” is used in conjunction with a numerical value/range, it modifies that value/range by extending the boundaries above and below the numerical value(s) set forth. In general, the term “about” is used herein to modify a numerical value(s) above and below the stated value(s) by a variance of 20%.
As used herein, the phrase “increased activity” refers to any metabolic enhancement resulting in increased levels of enzyme in a host cell. As known to one of ordinary skill in the art, enzyme activity may be increased by increasing the level of transcription, either by modifying promoter function or by increasing gene copy number, increasing translational efficiency of an enzyme messenger RNA, e.g., by modifying ribosomal binding, or by increasing the stability of an enzyme protein, at which the half-life of the protein is increased, will lead to more enzyme molecules in the cell. All of these represent non-limiting examples of increasing the activity of an enzyme. (mRNA Processing and Metabolism: Methods and Protocols, Edited by Daniel R. Schoenberg, Humana Press Inc., Totowa, N.J.; 2004; ISBN 1-59259-750-5; Prokaryotic Gene Expression (1999) Baumberg, S., Oxford University Press, ISBN 0199636036; The Structure and Function of Plastids (2006) Wise, R. R. and Hoober J. K., Springer, ISBN 140203217X; The Biomedical Engineering Handbook (2000) Bronzino, J. D., Springer, ISBN 354066808X).
In one aspect the invention also provides nucleic acids, which are at least 60%, 70%, 80% 90% or 95% identical to the promoter nucleic acids disclosed therein and to the nucleic acids, which encode proteins, for example enzymes for ethanol formation or host cell enzymes involved in the conversion or formation of acetyl CoA, acetaldehyde or pyruvate. The invention also provides amino acid sequences for enzymes for ethanol formation or host cell enzymes involved in the conversion or formation of acetyl-CoA, acetaldehyde or pyruvate or for formation of reserve compounds, which are at least 60%, 70%, 80% 90% or 95% identical to the amino acid sequences disclosed therein.
The percentage of identity of two nucleic acid sequences or two amino acid sequences can be determined using the algorithm of Thompson et al. (CLUSTALW, 1994 Nucleic Acid Research 22: 4673-4, 680). A nucleotide sequence or an amino acid sequence can also be used as a so-called “query sequence” to perform searches against public nucleic acid or protein sequence databases in order, for example, to identify further unknown homologous genes, which can also be used in embodiments of this invention. In addition, any nucleic acid sequences or protein sequences disclosed in this patent application can also be used as a “query sequence” in order to identify yet unknown sequences in public databases, which can encode for example new enzymes, which could be useful in this invention. Such searches can be performed using the algorithm of Karlin and Altschul (1999 Proceedings of the National Academy of Sciences U.S.A. 87: 2,264 to 2,268), modified as in Karlin and Altschul (1993 Proceedings of the National Academy of Sciences U.S.A. 90: 5,873 to 5,877). Such an algorithm is incorporated in the NBLAST and XBLAST programs of Altschul et al. (1999 Journal of Molecular Biology 215: 403 to 410). Suitable parameters for these database searches with these programs are, for example, a score of 100 and a word length of 12 for BLAST nucleotide searches as performed with the NBLAST program. BLAST protein searches are performed with the XBLAST program with a score of 50 and a word length of 3. Where gaps exist between two sequences, gapped BLAST is utilized as described in Altschul et al. (1997 Nucleic Acid Research, 25: 3,389 to 3,402).
Database entry numbers given in the following are for the CyanoBase, the genome database for cyanobacteria (http://bacteria.kazusa.or.jp/cyanobase/index.html); Yazukazu et al. “CyanoBase, the genome database for Synechocystis sp. Strain PCC6803: status for the year 2000”, Nucleic Acid Research, 2000, Vol. 18, page 72.
It is one object of embodiments of the invention to provide a metabolically enhanced host cell, which can be used for production of ethanol.
This object is reached by providing a metabolically enhanced host cell according to base claim 1. Further embodiments of the metabolically enhanced host cell, as well as constructs for producing the metabolically enhanced host cells and a method for producing ethanol using the metabolically enhanced host cells are subject matters of further claims.
Embodiment of metabolic knockout and/or overexpression of metabolic pathway enzymes
One aspect of the invention provides a metabolically enhanced photoautotrophic, ethanol producing host cell comprising:    at least two first metabolic enhancements reducing the enzymatic activity or affinity of at least two endogenous host cell enzymes involved in acetate and lactate fermentation,    the first metabolic enhancements resulting in an enhanced level of biosynthesis of acetaldehyde, pyruvate, acetyl-CoA or precursors thereof compared to the respective wild type host cell,    at least one second metabolic enhancement different from the first metabolic enhancement comprising an overexpressed enzyme for the formation of ethanol.
Acetaldehyde, pyruvate and acetyl-coA or their precursors are important metabolic intermediates for energy production in cells. In photoautotrophic cells, which use light, CO2, and water as a source of energy to produce carbohydrates via photosynthesis, acetaldehyde, pyruvate, acetyl-CoA and their precursors can be formed by conversion of organic molecules obtained via CO2 fixation in the Calvin-cycle, for example 3-phosphoglycerate. Pyruvate, acetyl-CoA and their precursors are important metabolic intermediates obtained e.g. by photosynthetic CO2 fixation in photoautotrophic cells. Acetaldehyde is a metabolic intermediate of the anoxygenic fermentation pathway in many photoautotrophic cells.
Precursors of pyruvate and acetyl-CoA are organic compounds, which can be converted into these important metabolic intermediates via the enzymatic action of enzymes of the photoautotrophic cell. For example the organic compounds 2-phosphoglycerate, 3-phosphoglycerate or phosphoenolpyruvate can be converted into pyruvate by enzymes of the glycolytic pathway in photoautotrophic cells.
The metabolically enhanced photoautotrophic ethanol producing host cell comprises at least two different metabolic enhancements, a first and a second metabolic enhancement. The first metabolic enhancement changes the enzymatic activity or affinity of at least two endogenous host cell enzymes involved in acetate and lactate fermentation, resulting in a higher level of biosynthesis of acetyl-CoA, acetaldehyde, pyruvate or precursors thereof. The endogenous host enzyme is already present in an unmodified wild type host cell and its activity or affinity is changed by the first metabolic enhancement in order to increase the level of biosynthesis of metabolic intermediates, which are also present in the wild type host cell and which can be used to form ethanol.
Furthermore the metabolically enhanced photoautotrophic ethanol producing host cell comprises a second metabolic enhancement in the form of at least one overexpressed enzyme, which can form ethanol, for example from the above-mentioned important metabolic intermediates. In a further embodiment the overexpressed enzyme for ethanol formation can catalyze the last step of ethanol formation leading to the final product ethanol. The overexpressed enzyme for ethanol formation can also catalyze the penultimate step of ethanol formation resulting in a metabolic intermediate, which can further be converted by another enzyme for ethanol formation into the final product ethanol.
The enzyme for ethanol formation can, for example, be an endogenous enzyme already present in a wild type photoautotrophic host cell, which is not metabolically enhanced. In this case the activity or affinity of the enzyme for ethanol formation can be enhanced by the second metabolic enhancement, for example by metabolic engineering or random mutagenesis. This can, for example, be done by metabolically modifying the amino acid sequence of the enzyme by site directed or random mutagenesis of the gene encoding this endogenous enzyme, thereby enhancing its activity for formation of ethanol. Another possibility is to increase the number of gene copies encoding for the enzyme in the host cell or simply by enhancing the rate of transcription of the gene already present in the wild type cell to increase the abundance of its messenger RNA in the second metabolic enhancement. This can be done for example by replacing or mutating the endogenous promoter controlling the transcription of the endogenous gene encoding the enzyme for ethanol formation.
Alternatively or additionally a heterologous enzyme for ethanol formation can be introduced into the host cell by the second metabolic enhancement, if that enzyme is not present in a metabolically unmodified wild type host cell. This can be done, for example, by introducing a construct, for example a DNA vector into the host cell including a heterologous gene encoding the overexpressed enzyme for ethanol formation. In the case that an endogenous enzyme for ethanol formation is already present in a photoautotrophic wild type host cell, the heterologous enzyme for ethanol formation can enhance the activity of the endogenous enzyme resulting in a higher rate of ethanol formation.
The enzymatic activity and the affinity of an enzyme for its substrate are important kinetic features. The enzymatic activity is given by the parameter Vmax, which reflects the maximal velocity of an enzymatic reaction occurring at high substrate concentrations when the enzyme is saturated with its substrate. The affinity is given by the Michaelis-Menten constant Km which is the substrate concentration required for an enzyme to reach one-half of its maximum velocity. In order to increase the enzymatic activity Vmax has to be increased, whereas for increasing the affinity Km has to be reduced. Regarding a further explanation of enzyme kinetics we refer to the chapter “enzyme kinetics” in the textbook “Biochemistry” by Donald Voet and Judith Voet (John Wiley & Sons, 1990, pages 335 to 340).
The higher level of biosynthesis of acetyl-CoA, acetaldehyde, pyruvate or precursors thereof results in a change of the flux of the acetyl-CoA, acetaldehyde, pyruvate or precursors thereof in the direction of the at least one overexpressed enzyme for ethanol formation so that formation of ethanol can be increased in comparison to a photoautotrophic ethanol producing host cell harboring only the second metabolic enhancement, but lacking the first metabolic enhancement.
Acetyl-CoA, acetaldehyde, pyruvate or precursors thereof are transient metabolic intermediates, which are often rapidly processed into other metabolites by the photoautotrophic host cell and therefore a change in the level of biosynthesis of these metabolic intermediates can be hard to detect in photoautotrophic host cells featuring the first metabolic enhancement but lacking the second metabolic enhancement.
A first metabolic enhancement therefore results in a higher level of biosynthesis of acetyl-CoA, acetaldehyde, pyruvate or precursors thereof compared to the respective wild type host cell, if after introduction of the second metabolic enhancement a higher level of ethanol formation can be detected in a cell harboring the first and second metabolic enhancement than in a cell only harboring the second metabolic enhancement but lacking the first metabolic enhancement. This even applies if a change in the level of biosynthesis of these metabolic intermediates could not be detected in the photoautotrophic host cell harboring the first metabolic enhancement but lacking the second metabolic enhancement in comparison to the respective wild-type photoautotrophic host cell, which does not harbor the first and second metabolic enhancement.
In particular, the metabolically enhanced photoautotrophic host cell can comprise more than two first metabolic enhancements and can also comprise more than one second metabolic enhancement. For example the first metabolic enhancements can comprise at least three metabolic enhancements, which are lactate dehydrogenase phosphotransacetylase and acetate kinase.
The inventors found out that by reducing the enzymatic affinity or activity of lactate dehydrogenase and an enzyme selected from phosphotransacetylase and acetate kinase the level of in particular pyruvate can be increased. Pyruvate is an important substrate for ethanologenic enzymes such as pyruvate decarboxylase, so that the pyruvate can be used for ethanol production.
The metabolically enhanced photoautotrophic host cell shows a high production of ethanol due to the fact that the ethanol forming enzyme is overexpressed due to the second metabolic enhancement leading to a high enzymatic activity for ethanol formation and that at the same time a higher level of biosynthesis of acetaldehyde, pyruvate, acetyl-CoA or their precursors is formed in the cells compared to the respective wild type cells due to the first metabolic enhancements. Acetaldehyde, pyruvate, acetyl-CoA or their precursors serve as substrates for the ethanol production. These metabolic intermediates can either be a direct substrate for a first overexpressed enzyme for the formation of ethanol or for another second overexpressed enzyme for ethanol formation, which then catalyzes the formation of a substrate for the first overexpressed enzyme for ethanol formation.
In yet a further embodiment of the host cell of the invention, the at least one overexpressed enzyme for the formation of ethanol is an alcohol dehydrogenase.
An alcohol dehydrogenase catalyzes the reduction of a substrate to ethanol. This reaction is normally dependent on the cofactor NADH. Alternatively there are alcohol dehydrogenases which are NADPH-dependent.
Furthermore, the alcohol dehydrogenase can be a thermaphilic alcohol dehydrogenase. Thermophilic alcohol dehydrogenase can, for example, be obtained from a host cell which can normally grow well at temperatures above 45° C. Thermophilic alcohol dehydrogenases can be more stable and probably more active at higher temperatures than alcohol dehydrogenases obtained from mesophilic host cells, which normally grow at temperatures below 45° C. One possible example for such a thermophilic alcohol dehydrogenase is the alcohol dehydrogenase AdhE obtained from the thermophilic cyanobacterium Thermosynechococcus sp. or from E. coil (see FIG. 44A for the nucleic acid sequence and FIG. 44B for the amino acid sequence of ThAdhE protein sequence BAC07780).
One possible substrate for alcohol dehydrogenase can be acetyl-CoA, which for example can be directly converted to ethanol by the above-mentioned alcohol dehydrogenase AdhE from Thermosynechococcus or E. coli. Overexpressing such an alcohol dehydrogenase in a metabolically enhanced host cell has the advantage that only one enzyme has to be overexpressed in order to enhance the level of ethanol production. In the case that the level of biosynthesis of acetyl-CoA of the host cell is increased due to overexpression of acetyl-coenzyme A forming enzymes and due to the reduction of enzymatic activity of acetyl-CoA converting enzymes, a high level of ethanol formation can result.
In a further embodiment of the invention, a metabolically enhanced host cell can be provided, which further comprises:
pyruvate decarboxylase converting pyruvate to acetaldehyde, wherein the alcohol dehydrogenase converts the acetaldehyde to ethanol.
In this case, the substrate for the alcohol dehydrogenase is provided by a further second overexpressed enzyme, for example pyruvate decarboxylase, which is introduced into the host cell via a further second metabolic enhancement. Due to the fact that the level of biosynthesis of pyruvate of the host cell is increased due to the above-mentioned enhancements of the pyruvate forming and converting enzymatic activities by way of the first metabolic enhancement, more acetaldehyde is formed via the enzymatic activity of pyruvate decarboxylase. Therefore there is an increased synthesis of acetaldehyde, which is then further converted by alcohol dehydrogenase, the first overexpressed enzyme for ethanol formation to ethanol resulting in a higher intracellular or extracellular ethanol level in the host cell. The alcohol dehydrogenase, as well as the pyruvate decarboxylase can be obtained from alcohol-fermenting organisms such as the bacteria Zymomonas mobilis, Zymobacter palmae or other prokaryots carrying genes encoding pyruvate decarboxylases with comparable or better enzymatic features as well as the yeast Saccharomyces cerevisiae or other eukaryotes carrying genes encoding pyruvate decarboxylases with comparable or better enzymatic features.
In another embodiment of the invention the metabolically enhanced host cell comprises two second metabolic enhancements, one comprising alcohol dehydrogenases Adh converting acetaldehyde into ethanol and another second metabolic enhancement comprising a CoA-dependent acetaldehyde dehydrogenase converting acetyl-CoA into acetaldehyde.
In yet a further embodiment of the invention the metabolically enhanced host cell harbors a pyruvate decarboxylase enzyme as the only second metabolic enhancement. Such a single second metabolic enhancement is particularly advantageous in metabolically enhanced host cells, which already have an endogenous alcohol dehydrogenase enzyme. The inventors surprisingly found that the activity of such an endogenous alcohol dehydrogenase enzyme can be high enough in order to convert all or almost all of the acetaldehyde formed by the overexpressed pyruvate decarboxylase enzyme into ethanol.
For example all cyanobacterial host cells harbor at least one endogenous alcohol dehydrogenase enzyme. A preferred example is the cyanobacterium Synechocystis in particular Synechocystis PCC6803 or nitrogen fixing cyanobacteria such as Nostoc/Anabaena spec. PCC7120 and Anabaena variabilis ATCC 29413.
In a further embodiment of the invention the metabolically enhanced photoautotrophic ethanol producing host cell is an aquatic organism. This aquatic organism can, for example, be a fresh water species living in lakes, rivers, streams or wetlands. Alternatively the aquatic organism can be a marine organism, which lives in salty water, for example oceans. The aquatic organism also can be a fresh water species, which shows a high tolerance for brackish water or even salt water. The inventors also found fresh water strains that can grow in marine media with the same growth rate as in fresh water media.
In a further embodiment the metabolically enhanced host cell is selected from a group consisting of: algae and bacteria.
Algae are a diverse group of simple plant-like organisms which include unicellular or multicellular forms. Algae are photosynthetically active organisms, in particular photoautotrophs, which produce organic compounds from inorganic molecules such as CO2 and water using light as an external source of energy.
Algae are considered to be eukaryotic organisms in particular protists. Protists are relatively simple eukaryotic organisms which are unicellular or multicellular without highly specialized tissues.
In particular, protist algae can include Chlorophytes, which are green algae, such as Ulva chlatrata, Rhodophytes, which are red algae or heterokontophytes, which are brown algae. A preferred green algal species is Chlorella. Another example of a green algae is Chlamydomonas, which are unicellular flagellates. A particular well known example of Chlamydomonas is Chlamydomonas reinhardtii, which is a motile single-celled green algae found in, for example, fresh water. Chlamydomonas reinhardtii is also known to produce minor amounts of ethanol via fermentation under dark conditions (Gfeller and Gibbs, Fermentative Metabolism of Chlamydomonas reinhardtii, Plant Psychology (1984) 75, pages 212 to 218).
In a further embodiment of the invention a metabolically enhanced photoautotrophic, ethanol producing host cell is provided, which comprises:
at least one first metabolic enhancement enhancing the enzymatic activity or affinity of the endogenous host cell enzymes selected from a group consisting of malic enzyme and malate dehydrogenase,
at least one second metabolic enhancement different from the at least two first metabolic enhancements comprising an overexpressed enzyme for the formation of ethanol,
the first and second metabolic enhancements resulting in an increased rate of ethanol production compared to the respective photoautotrophic, ethanol producing host cell harboring the second metabolic enhancement but lacking the first metabolic enhancements.
In the case that the enzymatic activity of malate dehydrogenase, an enzyme of the citric acid cycle and malic enzyme, an enzyme of the intermediate steps of metabolism is enhanced, for example by co-overexpression, malate dehydrogenase can stimulate the conversion of oxaloacetate to pyruvate via malate. Malate dehydrogenase catalyzes the conversion of oxaloacetate to malate using NADH:Oxaloacetate+NADH+H+→malate+NAD+
Malic enzyme catalyzes the conversion of malate into pyruvate using NADP+:malate+NADP+→pyzuvate+CO2+NADPH
Alternatively the enzymatic activity or affinity of only one of the enzymes malic enzyme or malate dehydrogenase can be enhanced, by for example overexpression.
In yet another embodiment of the invention a metabolically enhanced photoautotrophic, ethanol producing host cell is provided, which comprises:
at least one first metabolic enhancement enhancing the enzymatic activity or affinity of the endogenous host cell enzymes aldehyde dehydrogenase,
at least one second metabolic enhancement different from the at least one first metabolic enhancement comprising an overexpressed enzyme for the formation of ethanol,
the first and second metabolic enhancements resulting in an increased rate of ethanol production compared to the respective photoautotrophic, ethanol producing host cell harboring the second metabolic enhancement but lacking the first metabolic enhancement.
An enzyme of the fermentation pathway, which can be overexpressed is for example the aldehyde dehydrogenase enzyme, which can convert acetate to acetaldehyde and vice versa, thereby increasing the level of biosynthesis of acetaldehyde in the host cell. Alternatively also other aldehyde dehydrogenase enzymes could be overexpressed in order to increase the level of biosynthesis of acetaldehyde in the host cell.
In a further embodiment of the invention a metabolically enhanced photoautotrophic, ethanol producing host cell is provided, which comprises:
at least two first metabolic enhancements enhancing the enzymatic activity or affinity of the endogenous host cell enzymes phosphoketolase and phosphoacetyltransacetylase,
at least one second metabolic enhancement different from the at least two first metabolic enhancements comprising an overexpressed enzyme for the formation of ethanol,
the first and second metabolic enhancements resulting in an increased rate of ethanol production compared to the respective photoautotrophic, ethanol producing host cell harboring the second metabolic enhancement but lacking the first metabolic enhancements.
According to a further aspect of the invention the enzymatic activity or affinity of the enzyme phosphoketolase (EC 4.1.2.-, putative phosphoketolase in Synechocystis PCC 6803 slr 0453) is enhanced in a first metabolic enhancement in order to increase the level of biosynthesis of precursor molecules for the generation of acetyl-CoA and acetaldehyde. Phosphoketolase catalyses the formation of acetyl phosphate and glyceraldehyde 3-phosphate, a precursor of 3-phosphoglycerate from xylulose-5-phosphate which is an intermediate of the Calvin cycle. The concomitant enhancement of the enzymatic activity or affinity of the enzyme phosphoacetyltransacetylase, which catalyzes the interconversion of acetylphosphate and acetyl-CoA can enhance the level of, for example, acetyl-CoA and pyruvate. In particular the acetylphosphate produced by the overexpressed phosphoketolase can be further converted to acetyl-CoA via the enzymatic action of the overexpressed phosphoacetyltransacetylase. Without being bound by an theory, an enhanced level of acetyl-CoA might lead to a feed back effect and slow down the conversion of pyruvate to acetyl-CoA.
Any of the above mentioned enhancements, for example but not limiting the different ethanologenic enzymes for the second metabolic enhancement or the various different promoters, which are described in relation to the at least two first metabolic enhancements reducing the enzymatic activity or affinity of at least two endogenous host cell enzymes involved in acetate and lactate fermentation can also be used in conjunction with the enhancement of the enzymatic activity or affinity of malic enzyme and/or malate dehydrogenase, or aldehyde dehydrogenase or phosphoketolase and phosphoacetyltransacetylase.
Further in another embodiment of the invention, the metabolically enhanced host cell harboring any of the above disclosed first metabolic enhancements can also comprise overexpressed enzymes as a first metabolic enhancement or overexpressed ethanologenic enzymes for ethanol formation as a second metabolic enhancement, which are under the transcriptional control of various inducible or constitutive promoters, wherein the promoters are selected from a group consisting of:
rbcLS, ntcA, nblA, isiA, petJ, petE, sigB, lrtA, htpG, hspA, clpB1, hliB, ggpS, psbA2, psaA, nirA and crhC.
The promoters hspA, clpB1, and hliB can be induced by heat shock (raising the growth temperature of the host cell culture from 30° C. to 40° C.), cold shock (reducing the growth temperature of the cell culture from 30° C. to 20° C.), oxidative stress (for example by adding oxidants such as hydrogen peroxide to the culture), or osmotic stress (for example by increasing the salinity). The promoter sigB can be induced by stationary growth, heat shock, and osmotic stress. The promoters ntcA and nblA can be induced by decreasing the concentration of nitrogen in the growth medium and the promoters psaA and psbA2 can be induced by low light or high light conditions. The promoter htpG can be induced by osmotic stress and heat shock. The promoter crhC can be induced by cold shock. An increase in copper concentration can be used in order to induce the promoter petE, whereas the promoter petJ is induced by decreasing the copper concentration.
All the above promoter elements can be combined with any of the genes encoding any of the enzymes of the invention by using standard molecular cloning techniques. In particular the promoters, which can be used for the present invention include, but are not limited to:
(1) FIG. 45A depicts the nucleotide sequence of the petJ promoter (Synechocystis sp. PCC 6803) (petJ gene: sll1796 (encoding for cytochrome c553; induced expression under copper starvation);
Reference.:
J Biol Chem. 2004 February 20;279(8): 7229-33. Epub 2003 December.
The efficient functioning of photosynthesis and respiration in Synechocystis sp. PCC 6803 strictly requires the presence of either cytochrome c6 or plastocyanin.
Durán R V, Hervás M, De La Rosa M A, Navarro J A.
(2) FIG. 45B depicts the nucleotide sequence of the sigB promoter (Synechocystis sp. PCC 6803)
sigB gene: sll0306 (encoding for RNA polymerase group 2 sigma factor) induced expression after heat shock, in stationary growth phase/nitrogen starvation and darkness)
References:
Arch Microbial. 2006 October;186(4):273-86. Epub 2006 Jul. 26.    a. The heat shock response in the cyanobacterium Synechocystis sp. Strain PCC 6803 and regulation of gene expression by HrcA and SigB.    b. Singh A K, Summerfield T C, Li H, Sherman L A
FEBS Lett. 2003 November 20;554(3):357-62.    c. Antagonistic dark/light-induced SigB/SigD, group 2 sigma factors, expression through redox potential and their roles in cyanobacteria.    d. Imamura S, Asayama M, Takahashi H, Tanaka K, Takahashi H, Shirai M
J Biol Chem. 2006 February 3;281(5):2668-75. Epub 2005 Nov. 21.    e. Growth phase-dependent activation of nitrogen-related genes by a control network of group 1 and group 2 sigma factors in a cyanobacterium.    f. Imamura S, Tanaka K, Shirai M, Asayama M.
(3) FIG. 45C depicts the nucleotide sequence of the htpG promoter (Synechocystis sp. PCC 6803) htpG gene: s110430: (encoding for heat shock protein 90, molecular chaperone) induced expression after heat shock
Reference:
Plant Physiol. 1998 May;117(1):225-34.    g. Transcriptional and posttranscriptional control of mRNA from lrtA, a light-repressed transcript in Synechococcus sp. PCC 7002.    h. Samartzidou, H, Widger W R
(4) FIG. 45D shows the nucleotide sequence of the lrtA promoter (Synechocystis sp. PCC 6803) lrtA gene: sll0947 (encoding the light repressed protein A homolog induced expression after light to dark transition)
Reference:
Plant Physiol. 1998 May;117(1):225-34. Transcriptional and posttranscriptional control of mRNA from lrtA, a light-repressed transcript in Synechococcus sp. PCC 7002.    i. Samartzidou H, Widger W R
(5) the nucleotide sequence of the psbA2 promoter (Synechocystis sp. PCC 6803) (see FIG. 45E) psbA2 gene: slr1311 (encoding the photosystem II D1 protein) induced expression after dark to light transition    Reference:
Biochem Biophys Res Commun. 1999 February 5;255(1):47-53.    j. Light-dependent and rhythmic psbA transcripts in homologous/heterologous cyanobacterial cells.    k. Agrawal G K, Asayama M, Shirai M.
(6) FIG. 45F shows the nucleotide sequence of the rbcL promoter (Synechocystis sp. PCC 6803) rbcL gene: slr0009 (encoding the ribulose biphosphate carboxylase/oxygenase large subunit constitutive strong expression under continuous light conditions
Reference:
Plant Mol Biol. 1989 December;13(6):693-700
1. Influence of light on accumulation of photosynthesis-specific transcripts in the cyanobacterium Synechocystis 6803.
    m. Mohamed A, Janssen C.
(7) FIG. 45G depicts the nucleotide sequence of the psaA promoter (Synechocystis sp. PCC6803); PsaA gene: slr1834 (encoding P700 apoprotein subunit Ia) induced expression under low white light and orange light, to expression level under high light and red light, repressed in darkness
References:
Plant Cell Physiol. 2005 September;46(9):1484-93. Epub 2005 Jun. 24.
Regulation of photosystem I reaction center genes in Synechocystis sp. strain PCC 6803 during Light acclimation.
Herranen M, Tyystjärvi T, Aro E M.
Plant Cell Physiol. 2006 July;47(7):878-90. Epub 2006 May 16.
Characterization of high-light-responsive promoters of the psaAB genes in Synechocystis sp. PCC 6803.
Muramatsu M, Hihara Y.
(8) FIG. 45H shows the nucleotide sequence of the ggpS promoter (Synechocystis sp. PCC6803); ggpS gene: sll1566 (encoding glucosylglycerolphosphate synthase) induced expression after salt stress
Reference:
Plant Physiol. 2004 October;136(2):3290-300. Epub 2004 Sep. 10.
Gene expression profiling reflects physiological processes in salt acclimation of Synechocystis sp. strain PCC 6803.
Marin K, Kanesaki Y, Los D A, Murata N, Suzuki I, Hagemann M.
J. Bacteriol. 2002 June;184(11):2870-7.
Salt-dependent expression of glucosylglycerol-phosphate synthase, involved in osmolyte synthesis in the cyanobacterium Synechocystis sp. strain PCC 6803.
Marin K, Huckauf J, Fulda S, Hagemann M.
(9) FIG. 45I depicts the nucleotide sequence of the nirA promoter (Synechocystis sp. PCC6803); nirA gene: slr0898 (encoding ferredoxin-nitrite reductase) induced expression after transition from ammonia to nitrate.
Reference:
Appl. Environ Microbiol. 2005 October;71(10):5678-84.
Application of the Synechococcus nirA promoter to establish an inducible expression system for engineering the Synechocystis tocopherol pathway.
Qi Q, Hao M, Ng W O, Slater S C, Baszis S R, Weiss J D, Valentin H E.
J. Bacteriol. 1998 August;180(16):4080-8
cis-acting sequences required for NtcB-dependent, nitrite-responsive positive regulation of the nitrate assimilation operon in the cyanobacterium Synechococcus sp. strain PCC 7942.
Maeda S, Kawaguchi Y, Ohe T A, Omata T.
(10) FIG. 45J depicts the nucleotide sequence of the petE promoter (Anabaena sp. PCC7120); petE gene: all0258 (encoding plasocyanin precursor) induced expression at elevated copper concentrations
Reference:
Microbiology. 1994 May;140 (Pt 5):1151-9.
Cloning, sequencing and transcriptional studies of the genes for cytochrome c-553 and plastocyanin from Anabaena sp. PCC 7120.    n. Ghassemian M, Wong B, Ferreira F, Markley J L, Straus N A.
Proc Natl Acad Sci U S A. 2001 February 27;98(5):2729-34. Epub 2001 Feb. 20.
Expression of the Anabaena hetR gene from a copper-regulated promoter leads to heterocyst differentiation under repressing conditions.    o. Buikema W J, Haselkorn R.
(11) FIG. 45K shows the nucleotide sequence of the hspA promoter (Synechocystis sp. PCC6803); hspA gene: sll1514 16.6 kDa small heat shock protein, molecular chaperone
multi-stress responsible promoter (heat, cold, salt and oxidative stress)
Reference:
Curr Microbiol. 2004 September;49(3):192-8.
Expression of the heat shock gene hsp16.6 and promoter analysis in the cyanobacterium, Synechocystis sp. PCC 6803.
Fang F, Barnum S R.
J Exp Bot. 2006;57(7):1573-8. Epub 2006 Mar. 30.
The heat shock response of Synechocystis sp. PCC 6803 analyzed by transcriptomics and proteomics.
Suzuki I, Simon W J , Slabas A R.
(12) FIG. 45L depicts the nucleotide sequence of the hliB promoter (Synechocystis sp. PCC6803); hliB gene: ssr2595: high light-inducible polypeptide HliB, CAB/ELIP/HLIP superfamily multi-stress responsible promoter (heat, cold, salt and oxidative stress)
Reference:
J Biol Chem. 2001 January 5;276(1):306-14.
The high light-inducible polypeptides in Synechocystis PCC6803. Expression and function in high light.
He Q, Dolganov N, Bjorkman O, Grossman A R.
Arch Microbiol. 2007 April;187(4):337-42. Epub 2007 Feb. 10.
The response regulator RpaB binds the high light regulatory 1 sequence upstream of the high-light-inducible hliB gene from the cyanobacterium Synechocystis PCC 6803.
Kappell A D, van Waasbergen L G.
(13) FIG. 45M shows the nucleotide sequence of the clpB1 promoter (Synechocystis sp. PCC6803); clpB1 gene: slr1641: ATP-dependent Clp protease, Hsp 100, ATP-binding subunit ClpB
multi-stress responsible promoter (heat, cold, salt and oxidative stress)
Reference:
Microbiology. 2004 May;150(Pt 5):1271-81.
Effects of high light on transcripts of stress-associated genes for the cyanobacteria Synechocystis sp. PCC 6803 and Prochlorococcus MED4 and MIT9313.
Mary I, Tu C J, Grossman A, Vaulot D.
J Exp Bot. 2006;57(7):1573-8. Epub 2006 Mar. 30.
The heat shock response of Synechocystis sp. PCC 6803 analysed by transcriptomics and proteomics.
Suzuki I, Simon W J, Slabas A R.